Evidence for significantly enhancing reduction of Azo dyes in Escherichia coli by expressed cytoplasmic Azoreductase (AzoA) of Enterococcus faecalis

Abstract

Although cytoplasmic azoreductases have been purified and characterized from various bacteria, little evidence demonstrating that these azoreductases are directly involved in azo dye reduction in vivo is known. In order to evaluate the contribution of the enzyme to azo dye reduction in vivo, experiments were conducted to determine the effect of a recombinant cytoplasmic azoreductase (AzoA) from Enterococcus faecalis expressed in Escherichia coli on the rate of metabolism of Methyl Red, Ponceau BS and Orange II. The intact cells that contained IPTG induced AzoA had a higher rate of dye reduction with increases of 2 (Methyl Red), 4 (Ponceau BS) and 2.6 (Orange II)-fold compared to noninduced cells, respectively. Metabolites of Methyl Red isolated from induced cultures were identified as N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid through liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) analyses. In conclusion, our data demonstrate that AzoA from Ent. faecalis is capable of increasing the reduction of azo dyes in intact E. coli cells and that cytoplasmic azoreductase is involved in bacterial dye degradation in vivo.

Figure 1

Effect of different concentrations of NADH on the anaerobic reduction of Methyl Red. The concentration of Methyl Red was about 25 μM and about 200 mg (cell dry weight) L⁻¹ were added for both IPTG induced cell (triangles) and noninduced cell (circles), respectively. Decolorization was determined by measuring the decrease in optical density at 430 nm. The additional NADH was added to final concentrations of 0 (A), 0.2 (B), 0.5 (C), 1 (D), and 2 mM (E). Data were presented by the averages from triplicate incubations. Minor detectable reduction of the dyes was found in the control samples without cells in PBS buffer (pH 7.2) plus NADH was deducted from the data above as well.

Figure 2

Effect of different cell dry weight on the activity of anaerobic reduction of Methyl Red for both IPTG induced cell (triangles) and noninduced cell (circles). Decolorization was determined by measuring the decrease in optical density at 430 nm. Data were presented by the averages from triplicate incubations.

Figure 3

Product ion spectra of the metabolites from Methyl Red and standards: (A) metabolite eluting at 4.0 min, (B) N,N-dimethyl-p-phenylenediamine standard eluting at 4.0 min, (C) metabolite eluting at 25.2 min, (D) 2-aminobenzoic acid standard eluting at 25.2 min.

Figure 4

Proposed model for enhancing reduction of Methyl Red by E. coli containing azoreductase gene from Ent. faecalis with additional NADH. A, the situation without additional NADH, partial azoreductase cannot work for the lack of NADH; B, the situation with additional NADH. The schematic representation is not base on the proportion of the molecules.